Astroviruses have been long known to be a significant cause of gastroenteritis in children and in the young of many animal species (Méndez and Arias, 2013). The novel HAstVs have been isolated from stools, although a clear correlation with gastroenteritis is not established yet. They have been implicated, however, as agents of encephalitis in immunocompromised patients. In a fatal case of progressive encephalitis an astrovirus belonging to a recently discovered VA/HMO clade was identified in the brain of a 42-year-old man with chronic lymphocytic leukemia (Naccache et al., 2015), and an astrovirus phylogenetically related to ovine, mink, and bat astroviruses was reported to be the etiological agent of encephalitis in a 15-year-old boy with agammaglobulinemia (Quan et al., 2015). In another case report, the astrovirus HAstV-VA1/HMO-C-UK1 was identified in the brain and cerebrospinal fluid of an 18-month-old boy with encephalopathy, indicating that VA1/HMO-C viruses, unlike the classic astrovirus HAstV-1-8 serotypes, can be neuropathic (Brown et al., 2015). In an additional report, a child with severe primary combined immunodeficiency died with a disseminated viral infection, and astroviral RNA was detected in the brain and different other organs, while immunochemistry confirmed infection of gastrointestinal tissues (Wunderli et al., 2011). Similarly, astroviruses have been detected in brain tissue from minks with the neurological shaking syndrome (Blomstrom et al., 2010) and from a young adult crossbreed steer with acute onset of neurologic disease (Li et al., 2013). In avian species, astroviruses produce not only enteritis, but also more diverse pathologies such as hepatitis, poult enteritis mortality syndrome, and nephritis (Méndez and Arias, 2013; Liu et al., 2014).

The 5′-most ORFs 1a and 1b code for two nonstructural polyproteins (nsp’s). Nsp1a includes sequences encoded only in ORF1a, while nsp1ab includes sequences derived from both ORF1a and 1b. Protein nsp1ab is produced by a – 1 translational frameshift mechanism. There is an overlap of 10–148 encoding nt in the genome of mammalian viruses, and between 10 and 45 nt in avian viruses between ORFs 1a and 1b. This overlapping region contains a conserved heptameric sequence (AAAAAAC) followed by a potential stem-loop structure, motifs that are both essential for the frameshift and the synthesis of the viral polymerase encoded in ORF1b (Jiang et al., 1993; Marczinke et al., 1994). ORF2 encodes the precursor of the virus capsid polypeptides. A short predicted ORF X of 91–122 codons, conserved in all HAstV and some other mammalian astroviruses, is present in a + 1 reading frame within ORF 2, although there is no evidence of a protein derived from this ORF (Firth and Atkins, 2010). The capsid polyprotein precursor is synthesized from a subgenomic RNA (sgRNA) of about 2.4 kb in size (Monroe et al., 1993) that might be transcribed by the recognition of a cis-element that acts as a promoter on the antigenomic RNA (agRNA) (Méndez and Arias, 2013).

HAstV-1 and -8 are internalized into the cell by clathrin-dependent endocytosis, and the half-time for release of the genomic RNA (gRNA) into the cytoplasm for HAstV-8 is around 130 min (Donelli et al., 1992; Méndez et al., 2014). Drugs that disrupt endosome acidification and actin filament polymerization, as well as others that reduce the presence of cholesterol in the cell membrane decrease the infectivity of HAstV-8. Furthermore, in cells where the expression of Rab 7 is down regulated, the infectivity of HAstV-8 is also reduced. Altogether, these data support the notion that during cell entry astroviruses arrive to late endosomes, where the viral genome could exit into the cytosol (Méndez et al., 2014). During virus entry cellular signaling pathways are activated; in particular, it has been reported that astrovirus induces the transient phosphorylation of ERK1/2, independently of replication. Inhibitors of ERK affect viral protein and RNA synthesis with the consequent reduction of viral progeny (Moser and Schultz-Cherry, 2008). PI3K was also shown to reduce HAstV-1 infectivity, independently of MAPK, probably during cell entry (Tange et al., 2013).

Based on the transcription initiation site determined for the sgRNA in HAstV-1 and HAstV-2, ORF1b and ORF2 overlap in 8 nt; however, the length of this overlapping may vary and is not present in some viruses. The highly conserved sequence of around 40 nt upstream of the ORF2 start codon has been suggested to be part of the promoter for synthesis of the sgRNA. This conserved sequence shows partial identity with the 5′ end of the gRNA, suggesting that it has an important role for the synthesis of both gRNA and sgRNA (Méndez and Arias, 2013).

The structural proteins of the virus are translated from the sgRNA as a primary product of 87–90 kDa, named VP90 (Fig. 4.2.2B). The mechanism of translation of the sgRNA is not known, but it could depend on VPg (Fuentes et al., 2012). VP90 contains two domains classified by their sequence identity: a highly conserved amino-terminal domain (residues 1–415) and a highly divergent domain (residues 416 to the carboxy terminus) (Méndez and Arias, 2013; Mendez-Toss et al., 2000). The amino-terminal region contains a region of basic amino acids predicted to interact with the viral genome and a region proposed to form the capsid core (Méndez and Arias, 2013). The region between amino acids 416 and 647 are thought to form the spikes of the virion (Dong et al., 2011; Krishna, 2005), while the carboxy-terminal region of the hypervariable domain contains an acidic region that includes several potential sites for processing by cellular caspases (Méndez and Arias, 2013; Dong et al., 2011). Three proteins of 25, 27, and 34 kDa represent the final trypsin cleavage products present in the capsid of the mature HAstV-8 virion (Méndez and Arias, 2013).

As part of their replication cycle, viruses subvert intracellular membranes where viral and host factors cooperatively generate distinct organelle-like structures that serve as platform to form the replication complex of the virus (Paul and Bartenschlager, 2013). Recently, the cellular proteins present in membranes to which astroviral proteins and RNA associate were determined by LC-MS/MS. Functional analysis of the protein–protein interaction network showed some biological processes that were enriched in these membranes, such as gluconeogenesis, fatty acid beta-oxidation, fatty acid synthesis, long chain fatty acid synthesis and tricarboxylic acid cycle (Murillo et al., 2015). These findings are consistent with the fact that modification of the lipid metabolism is emerging as a landmark of the infection of positive-sense-viruses (Paul and Bartenschlager, 2013). Although it is not known if the ubiquitin/proteasome system contributes to cell membrane remodelling, it is also required for astrovirus replication; proteasomal inhibitors and knockdown of ubiquitin expression produce a reduction in viral protein synthesis, and gRNA and viral progeny production (Casorla et al., in preparation).

In astrovirus-infected Caco-2 cells, VP90 assembles intracellularly into viral particles, and is then processed by cellular caspases to yield viruses containing VP70 in a process that might involve at least three intermediate cleavage products of 82, 78, and 75 kDa (Méndez et al., 2004). Astrovirus infection induces apoptosis (Méndez et al., 2004; Guix et al., 2004a), and down regulation of the expression of caspases 3 and 9 blocks the processing of HAstV-8 VP90 to VP70 at the sequence motif TYVD657 (Baños-Lara and Méndez, 2010). The cleavage of VP90–VP70 is associated with release of the virus from the cell through a mechanism that does not involve cell lysis (Méndez et al., 2004; Baños-Lara and Méndez, 2010). All extracellular virions grown in cells in the absence of trypsin are composed by VP70, and to be fully infective these viral particles have to be proteolytically processed to yield the mature virions composed of three capsid proteins (Bass and Qiu, 2000; Méndez et al., 2002).

To overcome this problem, Velázquez-Moctezuma et al. (2012) developed a reverse genetic system using the highly transfectable hepatoma Huh7.5.1 cells to pack the virus. Although these cells were infected with a 100-fold lower efficiency than Caco-2 cells, the yield of infectious virus in both cells was similar, and in Huh7.5.1 cells the VP90 precursor of the astrovirus capsid polypeptides was found to be efficiently processed. Importantly, virus titers near to 10⁸ infectious units per ml were obtained in Huh7.5.1 at 16–20 h after transfection with total RNA isolated from astrovirus-infected Caco-2 cells. On the other hand, virus titers close to 10⁶ were recovered by using in vitro transcribed RNA from a full-length cDNA HAstV-1 clone; this virus yield was about two log steps higher than that obtained in BHK-21 cells (Geigenmuller et al., 1997). The lower efficiency of virus production in Huh7.5.1 cells when in vitro synthesized RNA was compared to gRNA purified from infected cells, suggested that a factor was missing for the efficient translation or replication of the transfected, synthetic viral RNA (Velázquez-Moctezuma et al., 2012). The 5′-end modification of the viral RNA seemed to determine the specific infectivity of the RNA, since virus recovery was abolished when the total RNA isolated from infected cells was treated with a protease, while it increased when the in vitro transcribed RNA was capped. These observations are consistent with the more recently description of the existence of a VPg protein covalently bound to the 5′-end of the astrovirus gRNA, essential for virus replication (Fuentes et al., 2012). Using this reverse genetic system, amino acids relevant for the function of VPg were recently explored (Fuentes et al., 2012).